First Reports and Morphological and Molecular Characterization of Pratylenchus delattrei and Quinisulcius capitatus Associated with Chickpea in Ethiopia

Abstract Chickpea (Cicer arietinum L.) is classed among the most important leguminous crops of high economic value in Ethiopia. Two plant-parasitic nematode species, Pratylenchus delattrei and Quinisulcius capitatus, were recovered from chickpea-growing areas in Ethiopia and characterized using molecular and morphological data, including the first scanning electron microscopy data for P. delattrei. New sequences of D2-D3 of 28S, ITS rDNA and mtDNA COI genes have been obtained from these species, providing the first COI sequences for P. delattrei and Q. capitatus, with both species being found for the first time on chickpea in Ethiopia. Furthermore, Pratylenchus delattrei was recovered in Ethiopia for the first time. The information obtained about these nematodes will be crucial to developing effective nematode management plans for future chickpea production.

Chickpea (Cicer arietinum L.) is classed second among the most important leguminous grain crops after the common bean, and is grown throughout tropical, subtropical, and temperate regions (Singh et al., 2008;FAOSTAT, 2020). Ethiopia is the largest producer of chickpeas in Africa, contributing 60% of the continent's total production and ranking sixth internationally (Shiferaw et al., 2007;FAOSTAT, 2020;Fikre et al., 2020).
Chickpea is grown in Ethiopia for both domestic consumption and export purposes. It is also used to restore soil fertility as part of a crop rotation with wheat and teff (Dadi et al., 2005;Shiferaw et al., 2007;Fikre et al., 2020).
In Ethiopia, growers of chickpea experience different diseases and insect pests in their fields for which management methods are being implemented, and although plant-parasitic nematodes also represent an important chickpea pest, their importance is usually neglected due to local inabilities to recognize relevant symptoms and/or in identifying the associated species (Castillo et al., 2008;Abebe et al., 2015;Sikora et al., 2018). The root-lesion nematodes (RLN), Pratylenchus spp., are ranked as the third most damaging group of plant-parasitic nematodes in terms of economic loss to agricultural production after root-knot and cyst nematodes (Castillo and Vovlas, 2007;Jones et al., 2013). Pratylenchus is the most important genus that infects chickpea roots globally and reduces crop yields (Di Vito et al., 1992;Thompson et al., 2010;Reen et al., 2019;Behmand et al., 2022;Rostad et al., 2022), and various Pratylenchus species from chickpea roots and rhizospheres have been reported from countries in Asia, Africa, Europe, North America, South America, and Australia (Castillo et al., 2008;Sikora et al., 2018;Zwart et al., 2019). According to studies by Hollaway et al. (2000) and Behmand et al. (2018), in different parts of Turkey where chickpea is grown, chickpea crops are generally considered as being more susceptible to P. neglectus, P. penetrans, and P. thornei attack than field pea, fava bean, and lupin bean crops, but less vulnerable than wheat crops. In Australia, P. thornei and P. neglectus are known to cause substantial damage to chickpea production (Riley and Kelly, 2002;Hollaway et al., 2008;Thompson et al., 2010). Likewise, P. thornei has been reported to cause severe crop losses in Syria, Morocco, Tunisia, Algeria, India, and Spain (Di Vito et al., 1992;Di Vito et al., 1994;Castillo et al., 1996;Ali and Sharma, 2003).
The stunt nematode, Quinisulcius capitatus (Allen, 1955) Siddiqi, 1971(= Tylenchorhynchus capitatus Allen, 1955) is a polyphagous ectoparasite with a wide host range, common in leguminous crops , field peas in the USA (Upadhaya et al., 2018), and is commonly found parasitizing chickpea fields in Tunisia, Morocco, and Turkey (Di Vito et al., 1994;Ali and Sharma, 2003;Catillo et al., 2008). Quinisulcius species are also widely distributed throughout tomato, pepper, cabbage, and potato crops in many countries worldwide (Bafokuzara, 1996;Baimey et al., 2009;Geraert, 2011;Hussain et al., 2019). The correct identification of nematodes using the link between DNA sequences and morphological characters is crucial in avoiding species misidentification (Janssen et al., 2017a(Janssen et al., , 2017b, and therefore for the implementation of effective pest management strategies and control measures (Munawar et al., 2021). Nevertheless, in sub-Saharan Africa (SSA), where facilities for morphological and molecular characterizations are scarce, nematode identification has hitherto been limited to genus level (Powers et al., 2011;Coye et al., 2018). For example, in Ethiopia, despite the number of described species of Pratylenchus (Janssen et al., 2017b;Singh et al., 2018;Nguyen et al., 2019;Handoo et al., 2021) and Quinisulcius (Geraert, 2011;Hussain et al., 2019), only P. goodeyi from enset (Peregrine and Bridge, 1992) and P. zeae, P. brachyurus, and P. coffeae from maize have been identified to date (Abebe et al., 2015). This current study reports for the first time the presence of P. delattrei in Ethiopia, and in addition, it provides the first report of P. delattrei and Q. capitatus associated with chickpea. This study also characterizes these two species based on morphological features obtained from light microscope (LM) and scanning electron microscope (SEM), molecular information of ITS, 28S of rDNA and COI of mtDNA. Overall, the study provides a better understanding of nematodes as a potential concern in chickpea production in the country.

Materials and Methods
Sample collection and nematode extraction: Soil and root samples were collected from chickpea growing areas in Minjar, Adea'a, and Mesekan districts during the 2021 main growing season, located in central and southern parts of Ethiopia. Details regarding sample locality, altitude, GPS coordinators, and GenBank accession numbers are summarized in Table 1. From each sampling locality, 20 soil cores were taken in a zig-zag pattern from within the top 30 cm using a 3 cm diameter tube from the chickpea rhizosphere, mixed to obtain a 500 g soil sample. For each sample, 80 chickpea roots were collected and put in labelled plastic bags. Subsequently, both soil and root samples were taken to the Plant Disease Diagnostics laboratory at Jimma University and stored at 4 o C until nematode extraction (Barker et al., 1969). The nematodes were extracted from aliquots of 100 ml of soil and 10 g of roots by the modified Baermann tray method described by Hooper et al. (2005).
Morphological characterization: Morphological and morphometric data were recorded from both temporary and permanent slides. In order to link molecular data with morphological vouchers of individual nematodes, live nematodes were heat relaxed by quickly passing them over a flame and examined, photographed, and measured using an Olympus BX51 DIC Microscope (Olympus Optical, Tokyo, Japan), equipped with an HD Ultra camera. Subsequently, each specimen was recovered from the temporary slide for genomic DNA extraction. For permanent slides, the nematode suspensions were concentrated in a drop of water in an embryo glass dish, with a few drops of fixative (4% formalin, 1% glycerol (in water) in it. The nematodes were immediately heated in a microwave (700 watts) for about 4 sec and left at room temperature for 1 h at 4°C for 24 h. This was followed by gradually transferring to anhydrous glycerin, ready to be mounted on glass slides as described by Seinhorst (1959). Specimens for scanning electron microscopy (SEM) were fixed in Trump's fixative, washed in 0.1 M-phosphate buffer (pH = 7.5), dehydrated in a graded series of ethanol solutions, critical point dried with liquid CO 2 and mounted on stubs with carbon tabs (double conductive tapes), coated with 25 nm gold, and photographed with a JSM-840 EM (JEOL) at 12 kV .
Phylogenetic analysis: Resulting sequences were compared with other relevant sequences available in the GenBank. Multiple alignments of the different DNA sequences were made using MUSCLE with default parameters, followed by manual trimming of the poorly aligned ends using Geneious 2022.1. Phylogenetic trees were created by using MrBayes 3.2.6, adding Geneious with the GTR + I + G model. The Markov chains for generating phylogenetic trees were set at 1 × 106 generations, four runs, 20% burnin and sub-sampling frequency of 500 generations (Huelsenbeck and Ronquist, 2001).

Measurements
See Table 2.

Description
Females: Vermiform and slightly curved ventrally after heat-killing and fixation. Labial region continuous from the rest of the body and lip region with three annuli. Under SEM (Figs. 1 C,D), en face view showing an oval oral aperture surrounded by six inner labial sensilla, submedian segments fused to oral disc, corresponding to head pattern group 2 according to Corbett and Clark (1983). Stylet was well developed (16-18 μm long) with anteriorly directed rounded knobs. The areolation was only visible at tail level and lateral field with four incisures, with the outer two being entirely crenate, and the inner lines being finely striated. Rounded to oval-shaped metacorpus with short isthmus, pharyngeal gland overlapped ventrally. Excretory pore ws located slightly above pharyngo-intestinal junction. There was a vulva, a transverse slit in ventral view, and well developed post-vulval uterine sac. The tail had (27-30) annuli, subcylindrical, and with rounded to conical, smooth terminus.
Voucher material: Vouchers (two females) are available in the UGent Nematode Collection (slide UGnem-314) of the Nematology Research Unit, Department of Biology, Ghent University, Ghent, Belgium.  (Fig. 3B). Finally, two identical COI sequences have been generated for the first time for P. delattrei, and these sequences were in a poorly supported sister relationship (0.68 PP) with P. parazeae (Fig. 3C).

Description
Females: The body of females spiral, or become C shaped after heat relaxation. The lip region hemispherical, set off with necks, with having five to six annulations, strong stylet (17.8-19.7 μm), long, rounded basal knobs, lateral field with five incisures. Rounded median bulb with strongly developed central valves, slender isthmus surrounded by nerve ring and conspicuous rounded cardia. Deirids absent and excretory pores at level between anterior margin and the middle of the basal pharyngeal bulb. Protruding vulva lips and poorly developed round spermatheca. The tail terminus conoid, distinctly annulated, tail cylindrical, with distinct phasmid at the middle of the tail.  (Fig. 4A). Our ITS rDNA sequences also formed a maximally supported clade with seven identical Q. capitatus ITS sequences from Pakistan (MT703005-MT703011) (Fig. 4B). However, two Q. capitatus sequences from Canada (MW027537-MW027538) are only 83% similar and were in an unresolved position with Tylenchorhynchus and Q. curvus sequences (Fig.  4B). The three identical COI sequences are the first sequences for Q. capitatus, and these sequences showed a weakly supported sister relationship with Amplimerlinius icarus and Tylenchorhynchus (0.63 vs. 0.58 PP) (Fig. 4C).

Male: Not found
Remarks: The studied specimens are morphologically and morphometrically similar to the original description (Allen, 1955) and subsequent descriptions by Mekete et al. (2008), Munawar et al. (2021), and Iqbal et al. (2021), except for the slightly longer stylet compared to populations from coffee in  and longer tail compared to the Canadian population (41.0-50.0 vs. 31.3-40.4 µm) ( Table 3). All of the specimens have five incisures in the lateral field and also conoid, enlarged and striated terminus shape (Fig. 2), agreeing with genus Quinisulcius, which sets it apart from Tylenchorhynchus (5 vs. 3-4) according to the key of Hunt et al. (2012). As in the current study, males have rarely been found (Hopper, 1959;Siddiqi, 1971;Knobloch and Laughlin, 1973;Maqbool, 1982;Mekete et al., 2008;Geraert, 2011). However, Iqbal et al. (2021), described Q. capitatus male specimens from apple, tomato, maize, potato, cabbage, and onion in Pakistan. Quinisulcius capitatus is known to parasitize over 27 plants across all continents (North America, Central and South America, temperate parts of Europe, Africa, Asia, Australia, and New Zealand) (Munawar et al., 2021). In Africa, this species has been reported in Ethiopia from coffee (Mekete et al., 2008), soybean in South Africa (Mbatyoti et al., 2020), and tomato and carrot in Benin (Baimey et al., 2009). The Ethiopian Q. capitatus specimens formed a well-supported clade with the Pakistan populations in our D2-D3 of 28S and ITS rDNA, however, the tree topology to the Canadian Q. capitatus population was not resolved for both gene regions (Figs. 4A,B). This suggests that the Canadian populations may have been mislabelled.

Discussion
Using morphological and molecular data, P. delattrei was detected for the first time in chickpea, and for the first time in Ethiopia. Other RLN species, i.e., P. zeae, P. alleni, P. alkan, P. erzurumensis P. mulchandi, P. coffeae, P. thornei, P. neglectus, P. mediterraneus, P. penetrans, P. brachyurus, and P. minyus, have previously been reported from the root and rhizosphere of chickpea, and their associated damage to crops has been widely studied in different countries (Di Vito et al., 1992;Di Vito et al., 1994, Castillo et al., 1996Ali and Sharma, 2003;Castillo et al., 2008;Hollaway et al., 2008;Thompson et al., 2010;Sikora et al., 2018;Zwart et al., 2019;Behmand et al., 2022;Rostad Figure 4: Bayesian 50% majority rule consensus phylogeny of Quinisulcius capitatus from Ethiopia and related species on 28S (A) and (B) ITS of rDNA genes and (C) COI of mtDNA using a GTR model. Branch support is indicated with PP. The sequences from this study were marked by blue color and bold font.